Plants and seeds of corn variety I135168

ABSTRACT

According to the invention, there is provided seed and plants of the corn variety designated I135168. The invention thus relates to the plants, seeds and tissue cultures of the variety I135168, and to methods for producing a corn plant produced by crossing a corn plant of variety I135168 with itself or with another corn plant, such as a plant of another variety. The invention further relates to corn seeds and plants produced by crossing plants of variety I135168 with plants of another variety, such as another inbred line. The invention further relates to the inbred and hybrid genetic complements of plants of variety I135168.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of corn breeding.In particular, the invention relates to corn seed and plants of thevariety designated I135168, and derivatives and tissue cultures thereof.

2. Description of Related Art

The goal of field crop breeding is to combine various desirable traitsin a single variety/hybrid. Such desirable traits include greater yield,better stalks, better roots, resistance to insecticides, herbicides,pests, and disease, tolerance to heat and drought, reduced time to cropmaturity, better agronomic quality, higher nutritional value, anduniformity in germination times, stand establishment, growth rate,maturity, and fruit size.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant. A plant cross-pollinates if pollen comes to it from aflower on a different plant.

Corn plants (Zea mays L.) can be bred by both self-pollination andcross-pollination. Both types of pollination involve the corn plant'sflowers. Corn has separate male and female flowers on the same plant,located on the tassel and the ear, respectively. Natural pollinationoccurs in corn when wind blows pollen from the tassels to the silks thatprotrude from the tops of the ear shoot.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants produces a uniform population ofhybrid plants that are heterozygous for many gene loci. Conversely, across of two plants each heterozygous at a number of loci produces apopulation of hybrid plants that differ genetically and are not uniform.The resulting non-uniformity makes performance unpredictable.

The development of uniform corn plant hybrids requires the developmentof homozygous inbred plants, the crossing of these inbred plants, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionare examples of breeding methods used to develop inbred plants frombreeding populations. Those breeding methods combine the geneticbackgrounds from two or more inbred plants or various other broad-basedsources into breeding pools from which new inbred plants are developedby selfing and selection of desired phenotypes. The new inbreds arecrossed with other inbred plants and the hybrids from these crosses areevaluated to determine which of those have commercial potential.

North American farmers plant tens of millions of acres of corn at thepresent time and there are extensive national and internationalcommercial corn breeding programs. A continuing goal of these cornbreeding programs is to develop corn hybrids that are based on stableinbred plants and have one or more desirable characteristics. Toaccomplish this goal, the corn breeder must select and develop superiorinbred parental plants.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a corn plant of thevariety designated I135168. Also provided are corn plants having all thephysiological and morphological characteristics of the inbred cornvariety I135168. The inbred corn plant of the invention may furthercomprise, or have, a cytoplasmic or nuclear factor that is capable ofconferring male sterility or otherwise preventing self-pollination, suchas by self-incompatibility. Parts of the corn plant of the presentinvention are also provided, for example, pollen obtained from an inbredplant and an ovule of the inbred plant.

The invention also concerns seed of the inbred corn variety I135168. Theinbred corn seed of the invention may be provided as an essentiallyhomogeneous population of inbred corn seed of the variety designatedI135168. Essentially homogeneous populations of inbred seed aregenerally free from substantial numbers of other seed. Therefore, in thepractice of the present invention, inbred seed generally forms at leastabout 97% of the total seed. The population of inbred corn seed of theinvention may be particularly defined as being essentially free fromhybrid seed. The inbred seed population may be separately grown toprovide an essentially homogeneous population of inbred corn plantsdesignated I135168.

In another aspect of the invention, a plant of corn variety I135168comprising an added heritable trait is provided. The heritable trait maycomprise a genetic locus that is a dominant or recessive allele. In oneembodiment of the invention, a plant of corn variety I135168 comprisinga single locus conversion in particular is provided. In specificembodiments of the invention, an added genetic locus confers one or moretraits such as, for example, male sterility, herbicide tolerance, insectresistance, disease resistance, waxy starch, modified fatty acidmetabolism, modified phytic acid metabolism, modified carbohydratemetabolism and modified protein metabolism. The trait may be, forexample, conferred by a naturally occurring maize gene introduced intothe genome of the variety by backcrossing, a natural or inducedmutation, or a transgene introduced through genetic transformationtechniques. When introduced through transformation, a genetic locus maycomprise one or more transgenes integrated at a single chromosomallocation.

In yet another aspect of the invention, an inbred corn plant of thevariety designated I135168 is provided, wherein acytoplasmically-inherited trait has been introduced into said inbredplant. Such cytoplasmically-inherited traits are passed to progenythrough the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continue to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which must have therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

In another aspect of the invention, a tissue culture of regenerablecells of a plant of variety I135168 is provided. The tissue culture willpreferably be capable of regenerating plants capable of expressing allof the physiological and morphological characteristics of the variety,and of regenerating plants having substantially the same genotype asother plants of the variety. Examples of some of the physiological andmorphological characteristics of the variety I135168 includecharacteristics related to yield, maturity, and kernel quality, each ofwhich is specifically disclosed herein. The regenerable cells in suchtissue cultures will preferably be derived from embryos, meristematiccells, immature tassels, microspores, pollen, leaves, anthers, roots,root tips, silk, flowers, kernels, ears, cobs, husks, or stalks, or fromcallus or protoplasts derived from those tissues. Still further, thepresent invention provides corn plants regenerated from the tissuecultures of the invention, the plants having all the physiological andmorphological characteristics of variety I135168.

In yet another aspect of the invention, processes are provided forproducing corn seeds or plants, which processes generally comprisecrossing a first parent corn plant with a second parent corn plant,wherein at least one of the first or second parent corn plants is aplant of the variety designated I135168. These processes may be furtherexemplified as processes for preparing hybrid corn seed or plants,wherein a first inbred corn plant is crossed with a second corn plant ofa different, distinct variety to provide a hybrid that has, as one ofits parents, the inbred corn plant variety I135168. In these processes,crossing will result in the production of seed. The seed productionoccurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting, preferably in pollinating proximity, seeds of afirst and second parent corn plant, and preferably, seeds of a firstinbred corn plant and a second, distinct inbred corn plant. Where theplants are not in pollinating proximity, pollination can nevertheless beaccomplished by transferring a pollen or tassel bag from one plant tothe other as described below.

A second step comprises cultivating or growing the seeds of said firstand second parent corn plants into plants that bear flowers (corn bearsboth male flowers (tassels) and female flowers (silks) in separateanatomical structures on the same plant). A third step comprisespreventing self-pollination of the plants, i.e., preventing the silks ofa plant from being fertilized by any plant of the same variety,including the same plant. This is preferably done by emasculating themale flowers of the first or second parent corn plant, (i.e., treatingor manipulating the flowers so as to prevent pollen production, in orderto produce an emasculated parent corn plant). Self-incompatibilitysystems may also be used in some hybrid crops for the same purpose.Self-incompatible plants still shed viable pollen and can pollinateplants of other varieties but are incapable of pollinating themselves orother plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent corn plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent corn plants. The harvestedseed can be grown to produce a corn plant or hybrid corn plant.

The present invention also provides corn seed and plants produced by aprocess that comprises crossing a first parent corn plant with a secondparent corn plant, wherein at least one of the first or second parentcorn plants is a plant of the variety designated I135168. In oneembodiment of the invention, corn seed and plants produced by theprocess are first generation (F₁) hybrid corn seed and plants producedby crossing an inbred in accordance with the invention with another,distinct inbred. The present invention further contemplates seed of anF₁ hybrid corn plant. Therefore, certain exemplary embodiments of theinvention provide an F₁ hybrid corn plant and seed thereof.

In still yet another aspect of the invention, the genetic complement ofthe corn plant variety designated I135168 is provided. The phrase“genetic complement” is used to refer to the aggregate of nucleotidesequences, the expression of which sequences defines the phenotype of,in the present case, a corn plant, or a cell or tissue of that plant. Agenetic complement thus represents the genetic make up of an inbredcell, tissue or plant, and a hybrid genetic complement represents thegenetic make up of a hybrid cell, tissue or plant. The invention thusprovides corn plant cells that have a genetic complement in accordancewith the inbred corn plant cells disclosed herein, and plants, seeds anddiploid plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that variety I135168 could be identified by any of themany well known techniques such as, for example, Simple Sequence LengthPolymorphisms (SSLPs) (Williams et al., 1990), Randomly AmplifiedPolymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF),Sequence Characterized Amplified Regions (SCARs), Arbitrary PrimedPolymerase Chain Reaction (AP-PCR), Amplified Fragment LengthPolymorphisms (AFLPs) (EP 534 858, specifically incorporated herein byreference in its entirety), and Single Nucleotide Polymorphisms (SNPs)(Wang et al., 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by corn plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of an inbred corn plant of the invention with a haploidgenetic complement of a second corn plant, preferably, another, distinctinbred corn plant. In another aspect, the present invention provides acorn plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

In still yet another aspect, the present invention provides a method ofproducing an inbred corn plant derived from the corn variety I135168,the method comprising the steps of: (a) preparing a progeny plantderived from corn variety I135168, wherein said preparing comprisescrossing a plant of the corn variety I135168 with a second corn plant;(b) crossing the progeny plant with itself or a second plant to producea seed of a progeny plant of a subsequent generation; (c) growing aprogeny plant of a subsequent generation from said seed of a progenyplant of a subsequent generation and crossing the progeny plant of asubsequent generation with itself or a second plant; and (d) repeatingsteps (c) and (d) for an addition 3–10 generations to produce an inbredcorn plant derived from the corn variety I135168. In the method, it maybe desirable to select particular plants resulting from step (c) forcontinued crossing according to steps (b) and (c). By selecting plantshaving one or more desirable traits, an inbred corn plant derived fromthe corn variety I135168 is obtained which possesses some of thedesirable traits of corn variety I135168 as well potentially otherselected traits.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions of Plant Characteristics

Barren Plants: Plants that are barren, i.e., lack an ear with grain, orhave an ear with only a few scattered kernels.

Cg: Colletotrichum graminicola rating. Rating times 10 is approximatelyequal to percent total plant infection.

CLN: Corn Lethal Necrosis (combination of Maize Chlorotic Mottle Virusand Maize Dwarf Mosaic virus) rating: numerical ratings are based on aseverity scale where 1=most resistant to 9=susceptible.

Cn: Corynebacterium nebraskense rating. Rating times 10 is approximatelyequal to percent total plant infection.

Cz: Cercospora zeae-maydis rating. Rating times 10 is approximatelyequal to percent total plant infection.

Dgg: Diatraea grandiosella girdling rating (values are percent plantsgirdled and stalk lodged).

Dropped Ears: Ears that have fallen from the plant to the ground.

Dsp: Diabrotica species root ratings (1=least affected to 9=severepruning).

Ear-Attitude: The attitude or position of the ear at harvest scored asupright, horizontal, or pendant.

Ear-Cob Color: The color of the cob, scored as white, pink, red, orbrown.

Ear-Cob Diameter: The average diameter of the cob measured at themidpoint.

Ear-Cob Strength: A measure of mechanical strength of the cobs tobreakage, scored as strong or weak.

Ear-Diameter: The average diameter of the ear at its midpoint.

Ear-Dry Husk Color: The color of the husks at harvest scored as buff,red, or purple.

Ear-Fresh Husk Color: The color of the husks 1 to 2 weeks afterpollination scored as green, red, or purple.

Ear-Husk Bract: The length of an average husk leaf scored as short,medium, or long.

Ear-Husk Cover: The average distance from the tip of the ear to the tipof the husks. Minimum value no less than zero.

Ear-Husk Opening: An evaluation of husk tightness at harvest scored astight, intermediate, or open.

Ear-Length: The average length of the ear.

Ear-Number Per Stalk: The average number of ears per plant.

Ear-Shank Internodes: The average number of internodes on the ear shank.

Ear-Shank Length: The average length of the ear shank.

Ear-Shelling Percent: The average of the shelled grain weight divided bythe sum of the shelled grain weight and cob weight for a single ear.

Ear-Silk Color: The color of the silk observed 2 to 3 days after silkemergence scored as green-yellow, yellow, pink, red, or purple.

Ear-Taper (Shape): The taper or shape of the ear scored as conical,semi-conical, or cylindrical.

Ear-Weight: The average weight of an ear.

Early Stand: The percent of plants that emerge from the ground asdetermined in the early spring.

ER: Ear rot rating (values approximate percent ear rotted).

Final Stand Count: The number of plants just prior to harvest.

GDUs: Growing degree units which are calculated by the Barger Method,where the heat units for a 24-h period are calculated as GDUs=[(Maximumdaily temperature+Minimum daily temperature)/2]−50. The highest maximumdaily temperature used is 86° F. and the lowest minimum temperature usedis 50° F.

GDUs to Shed: The number of growing degree units (GDUs) or heat unitsrequired for an inbred line or hybrid to have approximately 50% of theplants shedding pollen as measured from time of planting. GDUs to shedis determined by summing the individual GDU daily values from plantingdate to the date of 50% pollen shed.

GDUs to Silk: The number of growing degree units for an inbred line orhybrid to have approximately 50% of the plants with silk emergence asmeasured from time of planting. GDUs to silk is determined by summingthe individual GDU daily values from planting date to the date of 50%silking.

Hc2: Helminthosporium carbonum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hc3: Helminthosporium carbonum race 3 rating. Rating times 10 isapproximately equal to percent total plant infection.

Hm: Helminthosporium maydis race 0 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht1: Helminthosporium turcicum race 1 rating. Rating times 10 isapproximately equal to percent total plant infection.

Ht2: Helminthosporium turcicum race 2 rating. Rating times 10 isapproximately equal to percent total plant infection.

HtG: Chlorotic-lesion type resistance. +=indicates the presence of Htchlorotic-lesion type resistance; −=indicates absence of Htchlorotic-lesion type resistance; and +/−=indicates segregation of Htchlorotic-lesion type resistance. Rating times 10 is approximately equalto percent total plant infection.

Kernel-Aleurone Color: The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel-Cap Color: The color of the kernel cap observed at dry stage,scored as white, lemon-yellow, yellow, or orange.

Kernel-Endosperm Color: The color of the endosperm scored as white, paleyellow, or yellow.

Kernel-Endosperm Type: The type of endosperm scored as normal, waxy, oropaque.

Kernel-Grade: The percent of kernels that are classified as rounds.

Kernel-Length: The average distance from the cap of the kernel to thepedicel.

Kernel-Number Per Row: The average number of kernels in a single row.

Kernel-Pericarp Color: The color of the pericarp scored as colorless,red-white crown, tan, bronze, brown, light red, cherry red, orvariegated.

Kernel-Row Direction: The direction of the kernel rows on the ear scoredas straight, slightly curved, spiral, or indistinct (scattered).

Kernel-Row Number: The average number of rows of kernels on a singleear.

Kernel-Side Color: The color of the kernel side observed at the drystage, scored as white, pale yellow, yellow, orange, red, or brown.

Kernel-Thickness: The distance across the narrow side of the kernel.

Kernel-Type: The type of kernel scored as dent, flint, or intermediate.

Kernel-Weight: The average weight of a predetermined number of kernels.

Kernel-Width: The distance across the flat side of the kernel.

Kz: Kabatiella zeae rating. Rating times 10 is approximately equal topercent total plant infection.

Leaf-Angle: Angle of the upper leaves to the stalk scored as upright (0to 30 degrees), intermediate (30 to 60 degrees), or 1ax (60 to 90degrees).

Leaf-Color: The color of the leaves 1 to 2 weeks after pollinationscored as light green, medium green, dark green, or very dark green.

Leaf-Length: The average length of the primary ear leaf.

Leaf-Longitudinal Creases: A rating of the number of longitudinalcreases on the leaf surface 1 to 2 weeks after pollination. Creases arescored as absent, few, or many.

Leaf-Marginal Waves: A rating of the waviness of the leaf margin 1 to 2weeks after pollination. Rated as none, few, or many.

Leaf-Number: The average number of leaves of a mature plant. Countingbegins with the cotyledonary leaf and ends with the flag leaf.

Leaf-Sheath Anthocyanin: A rating of the level of anthocyanin in theleaf sheath 1 to 2 weeks after pollination, scored as absent,basal-weak, basal-strong, weak or strong.

Leaf-Sheath Pubescence: A rating of the pubescence of the leaf sheath.Ratings are taken 1 to 2 weeks after pollination and scored as light,medium, or heavy.

Leaf-Width: The average width of the primary ear leaf measured at itswidest point.

LSS: Late season standability (values times 10 approximate percentplants lodged in disease evaluation plots).

Moisture: The moisture of the grain at harvest.

On1: Ostrinia nubilalis 1st brood rating (1=resistant to 9=susceptible).

On2: Ostrinia nubilalis 2nd brood rating (1=resistant to 9=susceptible).

Relative Maturity: A maturity rating based on regression analysis. Theregression analysis is developed by utilizing check hybrids and theirpreviously established day rating versus actual harvest moistures.Harvest moisture on the hybrid in question is determined and thatmoisture value is inserted into the regression equation to yield arelative maturity.

Root Lodging: Root lodging is the percentage of plants that root lodge.A plant is counted as root lodged if a portion of the plant leans fromthe vertical axis by approximately 30 degrees or more.

Seedling Color: Color of leaves at the 6 to 8 leaf stage.

Seedling Height: Plant height at the 6 to 8 leaf stage.

Seedling Vigor: A visual rating of the amount of vegetative growth on a1 to 9 scale, where 1 equals best. The score is taken when the averageentry in a trial is at the fifth leaf stage.

Selection Index: The selection index gives a single measure of hybrid'sworth based on information from multiple traits. One of the traits thatis almost always included is yield. Traits may be weighted according tothe level of importance assigned to them.

Sr: Sphacelotheca reiliana rating is actual percent infection.

Stalk-Anthocyanin: A rating of the amount of anthocyanin pigmentation inthe stalk. The stalk is rated 1 to 2 weeks after pollination as absent,basal-weak, basal-strong, weak, or strong.

Stalk-Brace Root Color: The color of the brace roots observed 1 to 2weeks after pollination as green, red, or purple.

Stalk-Diameter: The average diameter of the lowest visible internode ofthe stalk.

Stalk-Ear Height: The average height of the ear measured from the groundto the point of attachment of the ear shank of the top developed ear tothe stalk.

Stalk-Internode Direction: The direction of the stalk internode observedafter pollination as straight or zigzag.

Stalk-Internode Length: The average length of the internode above theprimary ear.

Stalk Lodging: The percentage of plants that did stalk lodge. Plants arecounted as stalk lodged if the plant is broken over or off below theear.

Stalk-Nodes With Brace Roots: The average number of nodes having braceroots per plant.

Stalk-Plant Height: The average height of the plant as measured from thesoil to the tip of the tassel.

Stalk-Tillers: The percent of plants that have tillers. A tiller isdefined as a secondary shoot that has developed as a tassel capable ofshedding pollen.

Staygreen: Staygreen is a measure of general plant health near the timeof black layer formation (physiological maturity). It is usuallyrecorded at the time the ear husks of most entries within a trial haveturned a mature color. Scoring is on a 1 to 9 basis where 1 equals best.

STR: Stalk rot rating (values represent severity rating of 1=25% ofinoculated internode rotted to 9=entire stalk rotted and collapsed).

SVC: Southeastern Virus Complex (combination of Maize Chlorotic DwarfVirus and Maize Dwarf Mosaic Virus) rating; numerical ratings are basedon a severity scale where 1=most resistant to 9=susceptible (1988reactions are largely Maize Dwarf Mosaic Virus reactions).

Tassel-Anther Color: The color of the anthers at 50% pollen shed scoredas green-yellow, yellow, pink, red, or purple.

Tassel-Attitude: The attitude of the tassel after pollination scored asopen or compact.

Tassel-Branch Angle: The angle of an average tassel branch to the mainstem of the tassel scored as upright (less than 30 degrees),intermediate (30 to 45 degrees), or 1ax (greater than 45 degrees).

Tassel-Branch Number: The average number of primary tassel branches.

Tassel-Glume Band: The closed anthocyanin band at the base of the glumescored as present or absent.

Tassel-Glume Color: The color of the glumes at 50% shed scored as green,red, or purple.

Tassel-Length: The length of the tassel measured from the base of thebottom tassel branch to the tassel tip.

Tassel-Peduncle Length: The average length of the tassel peduncle,measured from the base of the flag leaf to the base of the bottom tasselbranch.

Tassel-Pollen Shed: A visual rating of pollen shed determined by tappingthe tassel and observing the pollen flow of approximately five plantsper entry. Rated on a 1 to 9 scale where 9=sterile, 1=most pollen.

Tassel-Spike Length: The length of the spike measured from the base ofthe top tassel branch to the tassel tip.

Test Weight: Weight of the grain in pounds for a given volume (bushel)adjusted to 15.5% moisture.

Yield: Yield of grain at harvest adjusted to 15.5% moisture.

II. Other Definitions

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny back to one of the parents, for example, a first generationhybrid (F₁) with one of the parental genotypes of the F₁ hybrid.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

F₁ Hybrid: The first generation progeny of the cross of two plants.

Genetic Complement: An aggregate of nucleotide sequences, the expressionof which sequences defines the phenotype in corn plants, or componentsof plants including cells or tissue.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Isozymes: Detectable variants of an enzyme, the variants catalyzing thesame reaction(s) but differing from each other, e.g., in primarystructure and/or electrophoretic mobility. The differences betweenisozymes are under single gene, codominant control. Consequently,electrophoretic separation to produce band patterns can be equated todifferent alleles at the DNA level. Structural differences that do notalter charge cannot be detected by this method.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Genetic loci that contribute, at least inpart, certain numerically representable traits that are usuallycontinuously distributed.

Regeneration: The development of a plant from tissue culture.

SSR profile: A profile of simple sequence repeats used as geneticmarkers and scored by gel electrophoresis following PCR™ amplificationusing flanking oligonucleotide primers.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing wherein essentially allof the desired morphological and physiological characteristics of aninbred are recovered in addition to the characteristics conferred by thesingle locus transferred into the inbred via the backcrossing technique.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic sequence which has been introduced into the nuclearor chloroplast genome of a maize plant by a genetic transformationtechnique.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

III. Inbred Corn Plant I135168

A. Origin and Breeding History

Inbred plant I135168 was derived from a cross between the lines 17INI4and SYNBA2. The origin and breeding history of inbred plant I135168 canbe summarized as follows:

Summer The inbred line 17INI4 (a proprietary DEKALB Genetics 1997Corporation inbred) was grown in nursery rows G97:4–90 and 4–91 andcrossed to the inbred line SYNBA2 (a proprietary DEKALB GeneticsCorporation inbred), grown in nursery rows G97:4–7 and 4–8. Winter TheS0 seed was grown and self-pollinated in nursery row 1997–98 MX:513.Summer The S1 seed was grown and self-pollinated in nursery rows 1998G98:105–50 through G98:105–21. 80 ears were selected. Summer S2 earswere grown and self-pollinated. 4 ears were 1999 selected in rowG97:211–37. Winter S3 ears were grown ear-to-row and self-pollinated. 3ears 1999–2000 were selected in nursery row 99/94S2/2198. Summer S4 earswere grown ear-to-row and self-pollinated. 2 ears 2000 from nursery row00LF5:513–14 were selected and designated as coded corn variety I135168.Winter S5 ears were grown ear-to-row and self-pollinated. 4 ears 2000–01from nursery row HI-KI-K5LS:29–85 were selected. Summer S6 ears weregrown ear-to-row and self-pollinated. 10 ears 2001 were selected fromnursery rows 01LCB:129–20 through 01LCB:129–22. Winter S7 ears weregrown ear-to-row and self-pollinated. 9 ears 2001–02 were selected fromnursery rows RALS:30641 through 30655. An additional 11 ears wereselected from nursery rows K4LS2:14–58 through 14–32. Summer S8 earswere grown ear-to-row and self-pollinated. Final 2002 selection wascompleted in nursery rows 19–3 through 19–44. This selection consistedof bulking S9 ears.

Corn variety I135168 shows uniformity and stability within the limits ofenvironmental influence for the traits described hereinafter in Table 1.I135168 has been self-pollinated and ear-rowed a sufficient number ofgenerations with careful attention paid to uniformity of plant type toensure homozygosity and phenotypic stability. No variant traits havebeen observed or are expected in I135168.

Inbred corn plants can be reproduced by planting the seeds of the inbredcorn plant I135168, growing the resulting corn plants underself-pollinating or sib-pollinating conditions with adequate isolationusing standard techniques well known to an artisan skilled in theagricultural arts. Seeds can be harvested from such a plant usingstandard, well known procedures.

B. Phenotypic Description

In accordance with another aspect of the present invention, there isprovided a corn plant having the physiological and morphologicalcharacteristics of corn plant I135168. A description of thephysiological and morphological characteristics of corn plant I135168 ispresented in Table 1.

TABLE 1 Morphological Traits for I135168 and Selected VarietiesCHARACTERISTIC I135168 I855358 I130251 1. STALK Diameter (width) cm. 1.62.1 1.9 Anthocyanin Absent Weak Absent Brace Root Color Faint AbsentAbsent Nodes With Brace 1.0 0.4 1.5 Roots Internode Direction StraightStraight Zig-zag Internode Length cm. 16.1  12.4  16.0  2. LEAF ColorDark Green Green Dark Green Length cm. 61.0  63.3  63.8  Width cm. 6.67.7 7.7 Sheath Anthocyanin Absent Moderate Absent Sheath PubescenceLight Light Light Marginal Waves Many Moderate Many Longitudinal CreasesModerate Moderate None 3. TASSEL Length cm. 37.2  33.8  33.6  SpikeLength cm. 20.3  20.6  16.4  Peduncle Length cm. 10.6  5.1 8.7 BranchNumber 7.7 15.6  8.7 Anther Color Pink Green-Yellow Yellow Glume ColorLight Red Green Purple Glume Band Absent Absent Absent 4. EAR Silk ColorPink Green-Yellow Purple Number Per Stalk 1.0 1.0 1.0 Position(attitude) Pendent Upright Pendent Length cm. 11.5  10.5  12.3  ShapeSemi- Semi-Conical Semi- Conical Conical Diameter cm. 3.1 4.2 3.2 ShankLength cm. 12.6  10.1  10.4  Husk Bract Short Short Short Husk Cover cm.6.0 7.3 8.0 Husk Opening Very Tight Moderate Tight Husk Color FreshGreen Green Green Husk Color Dry Buff Buff Buff Cob Diameter cm. 1.7 2.41.8 Cob Color Red Red White Shelling Percent 87.4  87.4  84.8  5. KERNELRow Number 12.4  16.6  12.0  Number Per Row 19.0  18.9  21.6  RowDirection Straight — Straight Type Flint Dent Intermediate Cap ColorOrange Yellow Yellow- Orange Side Color Orange Deep Yellow Orange Length(depth) mm. 8.0 10.9  9.2 Width mm. 7.0 7.8 7.6 Thickness 4.0 4.9 4.8Endosperm Type Normal Normal Normal Endosperm Color Orange Yellow Orange*These are typical values. Values may vary due to environment. Othervalues that are substantially equivalent are also within the scope ofthe invention.

C. Deposit Information

Applicant has made a deposit of at least 2500 seeds of corn varietyI135168 with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209 USA, which was assignedATCC Accession No. PTA-8160. The seeds were deposited with the ATCC onJan. 19, 2007, and were taken from a deposit maintained by MonsantoCompany since prior to the filing date of this application. Access tothis deposit will be available during the pendency of the application tothe Commissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. The deposit will bemaintained in the ATCC Depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the patent, whichever is longer, and will bereplaced if it becomes nonviable during that period. Applicant does notwaive any infringement of their rights granted under this patent orunder the Plant Variety Protection Act (7 U.S.C. 2321 et seq.).

IV. Further Embodiments of the Invention

In certain further aspects, the invention provides plants modified toinclude at least a first desired heritable trait. Such plants may, inone embodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique.

Backcrossing methods can be used with the present invention to improveor introduce a trait into a variety. The term backcrossing as usedherein refers to the repeated crossing of a hybrid progeny back to oneof the parental corn plants. The parental corn plant which contributesthe locus or loci for the desired trait is termed the nonrecurrent ordonor parent. This terminology refers to the fact that the nonrecurrentparent is used one time in the backcross protocol and therefore does notrecur.

The parental corn plant to which the locus or loci from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman et al., 1995;Fehr, 1987; Sprague and Dudley, 1988). In a typical backcross protocol,the original parent of interest (recurrent parent) is crossed to asecond variety (nonrecurrent parent) that carries the genetic locus ofinterest to be transferred. The resulting progeny from this cross arethen crossed again to the recurrent parent and the process is repeateduntil a corn plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredlocus from the nonrecurrent parent. The backcross process may beaccelerated by the use of genetic markers, such as SSR, RFLP, SNP orAFLP markers to identify plants with the greatest genetic complementfrom the recurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto add or substitute one or more new traits in the original variety andprogeny therefrom. To accomplish this, a genetic locus of the recurrentparent is modified or substituted with the desired locus from thenonrecurrent parent, while retaining essentially all of the rest of thedesired genetic, and therefore the desired physiological andmorphological constitution of the original plant. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait to the plant. The exactbackcrossing protocol will depend on the characteristic or trait beingaltered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many traits have been identified that are not regularly selected for inthe development of a new variety but that can be improved bybackcrossing techniques. A genetic locus conferring the traits may ormay not be transgenic. Examples of such traits known to those of skillin the art include, but are not limited to, male sterility, waxy starch,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, male fertility and enhanced nutritionalquality. These genes are generally inherited through the nucleus, butmay be inherited through the cytoplasm. Some known exceptions to thisare genes for male sterility, some of which are inheritedcytoplasmically, but still act as a single locus trait.

Direct selection may be applied where a genetic locus acts as a dominanttrait. An example of a dominant trait is the herbicide resistance trait.For this selection process, the progeny of the initial cross are sprayedwith the herbicide prior to the backcrossing. The spraying eliminatesany plants which do not have the desired herbicide resistancecharacteristic, and only those plants which have the herbicideresistance gene are used in the subsequent backcross. This process isthen repeated for all additional backcross generations.

Many useful traits are those which are introduced by genetictransformation techniques. Methods for the genetic transformation ofcorn are known to those of skill in the art. For example, methods whichhave been described for the genetic transformation of corn includeelectroporation (U.S. Pat. No. 5,384,253), electrotransformation (U.S.Pat. No. 5,371,003), microprojectile bombardment (U.S. Pat. No.5,550,318; U.S. Pat. No. 5,736,369, U.S. Pat. No. 5,538,880; and PCTPublication WO 95/06128), Agrobacterium-mediated transformation (U.S.Pat. No. 5,591,616 and E.P. Publication EP672752), direct DNA uptaketransformation of protoplasts (Omirulleh et al., 1993) and siliconcarbide fiber-mediated transformation (U.S. Pat. No. 5,302,532 and U.S.Pat. No. 5,464,765).

It is understood to those of skill in the art that a transgene need notbe directly transformed into a plant, as techniques for the productionof stably transformed corn plants that pass single loci to progeny byMendelian inheritance is well known in the art. Such loci may thereforebe passed from parent plant to progeny plants by standard plant breedingtechniques that are well known in the art. Examples of traits that maybe introduced into a corn plant according to the invention are providedbelow.

A. Male Sterility

Examples of genes conferring male sterility include those disclosed inU.S. Pat. No. 3,861,709, U.S. Pat. No. 3,710,511, U.S. Pat. No.4,654,465, U.S. Pat. No. 5,625,132, and U.S. Pat. No. 4,727,219, each ofthe disclosures of which are specifically incorporated herein byreference in their entirety. The use of herbicide-inducible malesterility genes is described in U.S. Pat. No. 6,762,344. Male sterilitygenes can increase the efficiency with which hybrids are made, in thatthey eliminate the need to physically emasculate the corn plant used asa female in a given cross.

Where one desires to employ male-sterility systems with a corn plant inaccordance with the invention, it may be beneficial to also utilize oneor more male-fertility restorer genes. For example, where cytoplasmicmale sterility (CMS) is used, hybrid seed production requires threeinbred lines: (1) a cytoplasmically male-sterile line having a CMScytoplasm; (2) a fertile inbred with normal cytoplasm, which is isogenicwith the CMS line for nuclear genes (“maintainer line”); and (3) adistinct, fertile inbred with normal cytoplasm, carrying a fertilityrestoring gene (“restorer” line). The CMS line is propagated bypollination with the maintainer line, with all of the progeny being malesterile, as the CMS cytoplasm is derived from the female parent. Thesemale sterile plants can then be efficiently employed as the femaleparent in hybrid crosses with the restorer line, without the need forphysical emasculation of the male reproductive parts of the femaleparent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the corn plant is utilized, e.g.,for silage, but in most cases, the seeds will be deemed the mostvaluable portion of the crop, so fertility of the hybrids in these cropsmust be restored. Therefore, one aspect of the current inventionconcerns plants of the corn variety I135168 comprising a genetic locuscapable of restoring male fertility in an otherwise male-sterile plant.Examples of male-sterility genes and corresponding restorers which couldbe employed with the plants of the invention are well known to those ofskill in the art of plant breeding and are disclosed in, for instance,U.S. Pat. No. 5,530,191; U.S. Pat. No. 5,689,041; U.S. Pat. No.5,741,684; and U.S. Pat. No. 5,684,242, the disclosures of which areeach specifically incorporated herein by reference in their entirety.

B. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee et al., (1988); Gleen etal., (1992) and Miki et al., (1990).

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvl-3 phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase (bar) genes) may alsobe used. See, for example, U.S. Pat. No. 4,940,835 to Shah, et al.,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate resistance. Examples of specific EPSPS transformationevents conferring glyphosate resistance are provided by U.S. Pat. No.6,040,497.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyltransferase gene isprovided in European application No. 0 242 246 to Leemans et al. DeGreefet al., (1989), describe the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acct-S1, Acc1-S2 and Acct-S3 genes described byMarshall et al., (1992).

Genes are also known conferring resistance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibila et al., (1991), describe thetransformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (1992).

C. Waxy Starch

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC1 plant to determine which BC1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

D. Disease Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin et al., (1993) (tomato Pto gene for resistance toPseudomonas syringae pv.); and Mindrinos et al., (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Logemann et al., (1992), for example, disclose transgenic plantsexpressing a barley ribosome-inactivating gene have an increasedresistance to fungal disease.

E. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al., (1986), who disclosethe cloning and nucleotide sequence of a Bt δ-endotoxin gene. Moreover,DNA molecules encoding δ-endotoxin genes can be purchased from theAmerican Type Culture Collection, Manassas, Va., for example, under ATCCAccession Nos. 40098, 67136, 31995 and 31998. Another example is alectin. See, for example, Van Damme et al., (1994), who disclose thenucleotide sequences of several Clivia miniata mannose-binding lectingenes. A vitamin-binding protein may also be used, such as avidin. SeePCT application US93/06487, the contents of which are herebyincorporated by reference. This application teaches the use of avidinand avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., (1993)(nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al., (1990), of baculovirusexpression of cloned juvenile hormone esterase, an inactivator ofjuvenile hormone.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (1994), who described enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

F. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.,(1992). Various fatty acid desaturases have also been described, such asa Saccharomyces cerevisiae OLE1 gene encoding Δ9-fatty acid desaturase,an enzyme which forms the monounsaturated palmitoleic (16:1) and oleic(18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA(McDonough et al., 1992); a gene encoding a stearoyl-acyl carrierprotein delta-9 desaturase from castor (Fox et al. 1993); Δ6- andΔ12-desaturases from the cyanobacteria Synechocystis responsible for theconversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma)(Reddy et al. 1993); a gene from Arabidopsis thaliana that encodes anomega-3 desaturase (Arondel et al. 1992)); plant Δ9-desaturases (PCTApplication Publ. No. WO 91/13972) and soybean and Brassica Δ15desaturases (European Patent Application Publ. No. EP 0616644).

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. In corn, this, for example, could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for corn mutants characterized by lowlevels of phytic acid. See Raboy et al., (2000).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., (1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al., (1992)(production of transgenic plants that express Bacillus licheniformisα-amylase), Elliot et al., (1993) (nucleotide sequences of tomatoinvertase genes), Sergaard et al., (1993) (site-directed mutagenesis ofbarley α-amylase gene), and Fisher et al., (1993) (maize endospermstarch branching enzyme II). The Z10 gene encoding a 10 kD zein storageprotein from maize may also be used to alter the quantities of 10 kDZein in the cells relative to other components (Kirihara et al., 1988).

G. Origin and Breeding History of an Exemplary Introduced Trait

85DGD1 MLms is a conversion of 85DGD1 to cytoplasmic male sterility.85DGD1 MLms was derived using backcross methods. 85DGD1 (a proprietaryinbred of Monsanto Company) was used as the recurrent parent and MLms, agermplasm source carrying ML cytoplasmic sterility, was used as thenonrecurrent parent. The breeding history of the converted inbred 85DGD1MLms can be summarized as follows:

Hawaii Nurseries Planting Date Made up S–O: Female row 585 male Apr. 2,1992 row 500 Hawaii Nurseries Planting Date S–O was grown and plantswere Jul. 15, 1992 backcrossed times 85DGD1 (rows 444 ′ 443) HawaiiNurseries Planting Date Bulked seed of the BC1 was grown and Nov. 18,1992 backcrossed times 85DGD1 (rows V3–27 ′ V3–26) Hawaii NurseriesPlanting Date Bulked seed of the BC2 was grown and Apr. 02, 1993backcrossed times 85DGD1 (rows 37 ′ 36) Hawaii Nurseries Planting DateBulked seed of the BC3 was grown and Jul. 14, 1993 backcrossed times85DGD1 (rows 99 ′ 98) Hawaii Nurseries Planting Date Bulked seed of BC4was grown and Oct. 28, 1993 backorossed times 85DGD1 (rows KS- 63 ′KS-62) Summer 1994 A single ear of the BC5 was grown and backcrossedtimes 85DGD1 (MC94-822 ′ MC94-822-7) Winter 1994 Bulked seed of the BC6was grown and backcrossed times 85DGD1 (3Q-1 ′ 3Q-2) Summer 1995 Seed ofthe BC7 was bulked and named 85DGD1 MLms.

H. Illustrative Procedures for Introduction of a Desired Trait

As described above, techniques for the production of corn plants withadded traits are well known in the art (see, e.g., Poehlman et al.,1995; Fehr, 1987; Sprague and Dudley, 1988). A non-limiting example ofsuch a procedure one of skill in the art would use for preparation of acorn plant of I135168 comprising an added trait is as follows:

-   -   (a) crossing corn plant I135168 to a second (nonrecurrent) corn        plant comprising a locus to be converted in corn plant I135168;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to corn plant I135168; and    -   (d) repeating steps (b) and (c) until a plant of variety I135168        is obtained comprising the locus.

Following these steps, essentially any locus may be introduced into cornvariety I135168. For example, molecular techniques allow introduction ofany given locus, without the need for phenotypic screening of progenyduring the backcrossing steps.

PCR and Southern hybridization are two examples of molecular techniquesthat may be used for confirmation of the presence of a given locus andthus conversion of that locus. The techniques are carried out asfollows: Seeds of progeny plants are grown and DNA isolated from leaftissue (see Sambrook et al., 2001; Shure et al. 1983). Approximately onegram of leaf tissue is lyophilized overnight in 15 ml polypropylenetubes. Freeze-dried tissue is ground to a power in the tube using aglass rod. Powdered tissue is mixed thoroughly with 3 ml extractionbuffer (7.0 urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1%sarcosine). Tissue/buffer homogenate is extracted with 3 mlphenol/chloroform. The aqueous phase is separated by centrifugation, andprecipitated twice using 1/10 volume of 4.4 M ammonium acetate pH 5.2,and an equal volume of isopropanol. The precipitate is washed with 75%ethanol and resuspended in 100–500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA,pH 8.0). The DNA may then be screened as desired for presence of thelocus.

For PCR, two hundred to 1000 ng genomic DNA from the progeny plant beingscreened is added to a reaction mix containing 10 mM Tris-HCl pH 8.3,1.5 mM MgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 μM each dATP, dCTP,dGTP, dTTP, 20% glycerol, 2.5 units Taq DNA polymerase and 0.5 μM eachof forward and reverse DNA primers that span a segment of the locusbeing converted. The reaction is run in a thermal cycling machine 3minutes at 94 C, 39 repeats of the cycle 1 minute at 94 C, 1 minute at50 C, 30 seconds at 72 C, followed by 5 minutes at 72 C. Twenty μl ofeach reaction mix is run on a 3.5% NuSieve gel in TBE buffer (90 mMTris-borate, 2 mM EDTA) at 50V for two to four hours. The amplifiedfragment is detected using an agarose gel. Detection of an amplifiedfragment corresponding to the segment of the locus spanned by theprimers indicates the presence of the locus.

For Southern analysis, plant DNA is restricted, separated in an agarosegel and transferred to a Nylon filter in 10× SCP (20 SCP: 2M NaCl, 0.6 Mdisodium phosphate, 0.02 M disodium EDTA) according to standard methods(Southern, 1975). Locus DNA or RNA sequences are labeled, for example,radioactively with ³²P by random priming (Feinberg & Vogelstein, 1983).Filters are prehybridized in 6× SCP, 10% dextran sulfate, 2% sarcosine,and 500 μg/ml denatured salmon sperm DNA. The labeled probe isdenatured, hybridized to the filter and washed in 2× SCP, 1% SDS at 65°for 30 minutes and visualized by autoradiography using Kodak XAR5 film.Presence of the locus is indicated by detection of restriction fragmentsof the appropriate size.

V. Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated I135168. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. No. 5,538,880; andU.S. Pat. No. 5,550,318, each incorporated herein by reference in theirentirety). By way of example, a tissue culture comprising organs such astassels or anthers has been used to produce regenerated plants (U.S.Pat. No. 5,445,961 and U.S. Pat. No. 5,322,789; the disclosures of whichare incorporated herein by reference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than 2 or3 nuclei. Methods to determine the correct stage are well known to thoseskilled in the art and include mitramycin fluorescent staining (Pace etal., 1987), trypan blue (preferred) and acetocarmine squashing. Themid-uninucleate microspore stage has been found to be the developmentalstage most responsive to the subsequent methods disclosed to ultimatelyproduce plants.

Although microspore-containing plant organs such as tassels cangenerally be pretreated at any cold temperature below about 25° C., arange of 4 to 25° C. is preferred, and a range of 8 to 14° C. isparticularly preferred. Although other temperatures yield embryoids andregenerated plants, cold temperatures produce optimum response ratescompared to pretreatment at temperatures outside the preferred range.Response rate is measured as either the number of embryoids or thenumber of regenerated plants per number of microspores initiated inculture. Exemplary methods of microspore culture are disclosed in, forexample, U.S. Pat. No. 5,322,789 and U.S. Pat. No. 5,445,961, thedisclosures of which are specifically incorporated herein by reference.

Although not required, when tassels are employed as the plant organ, itis generally preferred to sterilize their surface. Following surfacesterilization of the tassels, for example, with a solution of calciumhypochloride, the anthers are removed from about 70 to 150 spikelets(small portions of the tassels) and placed in a preculture orpretreatment medium. Larger or smaller amounts can be used depending onthe number of anthers.

When one elects to employ tassels directly, tassels are preferablypretreated at a cold temperature for a predefined time, preferably at10° C. for about 4 days. After pretreatment of a whole tassel at a coldtemperature, dissected anthers are further pretreated in an environmentthat diverts microspores from their developmental pathway. The functionof the preculture medium is to switch the developmental program from oneof pollen development to that of embryoid/callus development. Anembodiment of such an environment in the form of a preculture mediumincludes a sugar alcohol, for example mannitol or sorbitol, inositol orthe like. An exemplary synergistic combination is the use of mannitol ata temperature of about 10° C. for a period ranging from about 10 to 14days. In a preferred embodiment, 3 ml of 0.3 M mannitol combined with 50mg/l of ascorbic acid, silver nitrate, and colchicine is used forincubation of anthers at 10° C. for between 10 and 14 days. Anotherembodiment is to substitute sorbitol for mannitol. The colchicineproduces chromosome doubling at this early stage. The chromosomedoubling agent is preferably only present at the preculture stage.

It is believed that the mannitol or other similar carbon structure orenvironmental stress induces starvation and functions to forcemicrospores to focus their energies on entering developmental stages.The cells are unable to use, for example, mannitol as a carbon source atthis stage. It is believed that these treatments confuse the cellscausing them to develop as embryoids and plants from microspores.Dramatic increases in development from these haploid cells, as high as25 embryoids in 10⁴ microspores, have resulted from using these methods.

To isolate microspores, an isolation media is preferred. An isolationmedia is used to separate microspores from the anther walls whilemaintaining their viability and embryogenic potential. An illustrativeembodiment of an isolation media includes a 6% sucrose or maltosesolution combined with an antioxidant such as 50 mg/l of ascorbic acid,0.1 mg/l biotin, and 400 mg/l of proline, combined with 10 mg/l ofnicotinic acid and 0.5 mg/l AgNO₃. In another embodiment, the biotin andproline are omitted.

An isolation media preferably has a higher antioxidant level where it isused to isolate microspores from a donor plant (a plant from which aplant composition containing a microspore is obtained) that is fieldgrown in contrast to greenhouse grown. A preferred level of ascorbicacid in an isolation medium is from about 50 mg/l to about 125 mg/l and,more preferably, from about 50 mg/l to about 100 mg/l.

One can find particular benefit in employing a support for themicrospores during culturing and subculturing. Any support thatmaintains the cells near the surface can be used. An illustrativeembodiment of a solid support is a TRANSWELL® culture dish. Anotherembodiment of a solid support for development of the microspores is abilayer plate wherein liquid media is on top of a solid base. Otherembodiments include a mesh or a millipore filter. Preferably, a solidsupport is a nylon mesh in the shape of a raft. A raft is defined as anapproximately circular support material which is capable of floatingslightly above the bottom of a tissue culture vessel, for example, apetri dish, of about a 60 or 100 mm size, although any other laboratorytissue culture vessel will suffice. In an illustrative embodiment, araft is about 55 mm in diameter.

Culturing isolated microspores on a solid support, for example, on a 10mm pore nylon raft floating on 2.2 ml of medium in a 60 mm petri dish,prevents microspores from sinking into the liquid medium and thusavoiding low oxygen tension. These types of cell supports enable theserial transfer of the nylon raft with its associatedmicrospore/embryoids ultimately to full strength medium containingactivated charcoal and solidified with, for example, GELRITE™(solidifying agent).

The liquid medium passes through the mesh while the microspores areretained and supported at the medium-air interface. The surface tensionof the liquid medium in the petri dish causes the raft to float. Theliquid is able to pass through the mesh; consequently, the microsporesstay on top. The mesh remains on top of the total volume of liquidmedium.

The culture vessels can be further defined as either (1) a bilayer 60 mmpetri plate wherein the bottom 2 ml of medium are solidified with 0.7%agarose overlaid with 1 mm of liquid containing the microspores; (2) anylon mesh raft wherein a wafer of nylon is floated on 1.2 ml of mediumand 1 ml of isolated microspores is pipetted on top; or (3) TRANSWELL®plates wherein isolated microspores are pipetted onto membrane insertswhich support the microspores at the surface of 2 ml of medium.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application 0 160 390, Greenand Rhodes (1982) and Duncan et al. (1985), Songstad et al. (1988), Raoet al. (1986), Conger et al. (1987), PCT Application WO 95/06128,Armstrong and Green, 1985; Gordon-Kanim et al., 1990, and U.S. Pat. No.5,736,369.

VI. Processes of Preparing Corn Plants and the Corn Plants Produced bySuch Crosses

The present invention provides processes of preparing novel corn plantsand corn plants produced by such processes. In accordance with such aprocess, a first parent corn plant may be crossed with a second parentcorn plant wherein at least one of the first and second corn plants isthe inbred corn plant I135168. One application of the process is in theproduction of F₁ hybrid plants. Another important aspect of this processis that it can be used for the development of novel inbred lines. Forexample, the inbred corn plant I135168 could be crossed to any secondplant, and the resulting hybrid progeny each selfed for about 5 to 7 ormore generations, thereby providing a large number of distinct,pure-breeding inbred lines. These inbred lines could then be crossedwith other inbred or non-inbred lines and the resulting hybrid progenyanalyzed for beneficial characteristics. In this way, novel inbred linesconferring desirable characteristics could be identified.

Corn plants (Zea mays L.) can be crossed by either natural or mechanicaltechniques. Natural pollination occurs in corn when wind blows pollenfrom the tassels to the silks that protrude from the tops of therecipient ears. Mechanical pollination can be effected either bycontrolling the types of pollen that can blow onto the silks or bypollinating by hand. In one embodiment, crossing comprises the steps of:

-   -   (a) planting in pollinating proximity seeds of a first and a        second parent corn plant, and preferably, seeds of a first        inbred corn plant and a second, distinct inbred corn plant;    -   (b) cultivating or growing the seeds of the first and second        parent corn plants into plants that bear flowers;    -   (c) emasculating flowers of either the first or second parent        corn plant, i.e., treating the flowers so as to prevent pollen        production, or alternatively, using as the female parent a male        sterile plant, thereby providing an emasculated parent corn        plant;    -   (d) allowing natural cross-pollination to occur between the        first and second parent corn plants;    -   (e) harvesting seeds produced on the emasculated parent corn        plant; and, where desired,    -   (f) growing the harvested seed into a corn plant, preferably, a        hybrid corn plant.

Parental plants are typically planted in pollinating proximity to eachother by planting the parental plants in alternating rows, in blocks orin any other convenient planting pattern. Where the parental plantsdiffer in timing of sexual maturity, it may be desired to plant theslower maturing plant first, thereby ensuring the availability of pollenfrom the male parent during the time at which silks on the female parentare receptive to pollen. Plants of both parental parents are cultivatedand allowed to grow until the time of flowering. Advantageously, duringthis growth stage, plants are in general treated with fertilizer and/orother agricultural chemicals as considered appropriate by the grower.

At the time of flowering, in the event that plant I135168 is employed asthe male parent, the tassels of the other parental plant are removedfrom all plants employed as the female parental plant to avoidself-pollination. The detasseling can be achieved manually but also canbe done by machine, if desired. Alternatively, when the female parentcorn plant comprises a cytoplasmic or nuclear gene conferring malesterility, detasseling may not be required. Additionally, a chemicalgametocide may be used to sterilize the male flowers of the femaleplant. In this case, the parent plants used as the male may either notbe treated with the chemical agent or may comprise a genetic factorwhich causes resistance to the emasculating effects of the chemicalagent. Gametocides affect processes or cells involved in thedevelopment, maturation or release of pollen. Plants treated with suchgametocides are rendered male sterile, but typically remain femalefertile. The use of chemical gametocides is described, for example, inU.S. Pat. No. 4,936,904, the disclosure of which is specificallyincorporated herein by reference in its entirety. Furthermore, the useof glyphosate herbicide to produce male sterile corn plants is disclosedin U.S. Pat. No. 6,762,344 and PCT Publication WO 98/44140.

Following emasculation, the plants are then typically allowed tocontinue to grow and natural cross-pollination occurs as a result of theaction of wind, which is normal in the pollination of grasses, includingcorn. As a result of the emasculation of the female parent plant, allthe pollen from the male parent plant is available for pollinationbecause tassels, and thereby pollen bearing flowering parts, have beenpreviously removed from all plants of the inbred plant being used as thefemale in the hybridization. Of course, during this hybridizationprocedure, the parental varieties are grown such that they are isolatedfrom other corn fields to minimize or prevent any accidentalcontamination of pollen from foreign sources. These isolation techniquesare well within the skill of those skilled in this art.

Both parental inbred plants of corn may be allowed to continue to growuntil maturity or the male rows may be destroyed after flowering iscomplete. Only the ears from the female inbred parental plants areharvested to obtain seeds of a novel F₁ hybrid. The novel F₁ hybrid seedproduced can then be planted in a subsequent growing season incommercial fields or, alternatively, advanced in breeding protocols forpurposes of developing novel inbred lines.

Alternatively, in another embodiment of the invention, both first andsecond parent corn plants can be from variety I135168. Thus, any cornplant produced using corn plant I135168 forms a part of the invention.As used herein, crossing can mean selfing, backcrossing, crossing toanother or the same inbred, crossing to populations, and the like. Allcorn plants produced using the inbred corn plant I135168 as a parentare, therefore, within the scope of this invention.

A. F₁ Hybrid Corn Plant and Seed Production

One beneficial use of the instant corn variety is in the production ofhybrid seed. Any time the inbred corn plant I135168 is crossed withanother, different, corn inbred, a first generation (F₁) corn hybridplant is produced. As such, an F₁ hybrid corn plant can be produced bycrossing I135168 with any second inbred maize plant. Essentially anyother corn plant can be used to produce a hybrid corn plant having cornplant I135168 as one parent. All that is required is that the secondplant be fertile, which corn plants naturally are, and that the plant isnot corn variety I135168.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

Corn has a diploid phase which means two conditions of a gene (twoalleles) occupy each locus (position on a chromosome). If the allelesare the same at a locus, there is said to be homozygosity. If they aredifferent, there is said to be heterozygosity. In a completely inbredplant, all loci are homozygous. Because many loci when homozygous aredeleterious to the plant, in particular leading to reduced vigor, lesskernels, weak and/or poor growth, production of inbred plants is anunpredictable and arduous process. Under some conditions, heterozygousadvantage at some loci effectively bars perpetuation of homozygosity.

A single cross hybrid corn variety is the cross of two inbred plants,each of which has a genotype which complements the genotype of theother. Typically, F₁ hybrids are more vigorous than their inbredparents. This hybrid vigor, or heterosis, is manifested in manypolygenic traits, including markedly improved yields, better stalks,better roots, better uniformity and better insect and diseaseresistance. In the development of hybrids only the F₁ hybrid plants aretypically sought. An F₁ single cross hybrid is produced when two inbredplants are crossed. A double cross hybrid is produced from four inbredplants crossed in pairs (A×B and C×D) and then the two F₁ hybrids arecrossed again (A×B)×(C×D).

Thousands of corn varieties are known to those of skill in the art, anyone of which could be crossed with corn plant I135168 to produce ahybrid plant. For example, the U.S. Patent & Trademark has issued morethan 300 utility patents for corn varieties. Estimates place the numberof different corn accessions in genebanks around the world at around50,000 (Chang, 1992). The Maize Genetics Cooperation Stock Center, whichis supported by the U.S. Department of Agriculture, has a totalcollection approaching 80,000 individually pedigreed samples(//w3.ag.uiuc.edu/maize-coop/mgc-info.html).

An example of an F₁ hybrid which has been produced with I135168 as aparent is the hybrid NB2701. Hybrid NB2701 was produced by crossinginbred corn plant I135168 as a male parent with a hybrid produced bycrossing the corn varieties I325350 and I918166 (U.S. patent applicationSer. Nos. 10/370,027 and 10/369,190, filed Feb. 19, 2003, thedisclosures of which are specifically incorporated herein by referencein the entirety).

When the inbred corn plant I135168 is crossed with another inbred plantto yield a hybrid, it can serve as either the maternal or paternalplant. For many crosses, the outcome is the same regardless of theassigned sex of the parental plants. However, due to increased seedyield and production characteristics, it may be desired to use oneparental plant as the maternal plant. Some plants produce tighter earhusks leading to more loss, for example due to rot. There can be delaysin silk formation which deleteriously affect timing of the reproductivecycle for a pair of parental inbreds. Seed coat characteristics can bepreferable in one plant. Pollen can be shed better by one plant. Othervariables can also affect preferred sexual assignment of a particularcross. The instant may variety may potentially be used as a male orfemale parent.

B. Development of Corn Varieties Using I135168

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing corn variety I135168 followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing corn variety I135168 with anysecond plant. In selecting such a second plant to cross for the purposeof developing novel inbred lines, it may be desired to choose thoseplants which either themselves exhibit one or more selected desirablecharacteristics or which exhibit the desired characteristic(s) when inhybrid combination. Examples of potentially desired characteristicsinclude greater yield, better stalks, better roots, resistance toinsecticides, herbicides, pests, and disease, tolerance to heat anddrought, reduced time to crop maturity, better agronomic quality, highernutritional value, and uniformity in germination times, standestablishment, growth rate, maturity, and fruit size.

Once initial crosses have been made with corn variety I135168,inbreeding takes place to produce new inbred varieties. Inbreedingrequires manipulation by human breeders. Even in the extremely unlikelyevent inbreeding rather than crossbreeding occurred in natural corn,achievement of complete inbreeding cannot be expected in nature due towell known deleterious effects of homozygosity and the large number ofgenerations the plant would have to breed in isolation. The reason forthe breeder to create inbred plants is to have a known reservoir ofgenes whose gametic transmission is predictable.

The pedigree breeding method involves crossing two genotypes. Eachgenotype can have one or more desirable characteristics lacking in theother; or, each genotype can complement the other. If the two originalparental genotypes do not provide all of the desired characteristics,other genotypes can be included in the breeding population. Superiorplants that are the products of these crosses are selfed and selected insuccessive generations. Each succeeding generation becomes morehomogeneous as a result of self-pollination and selection. Typically,this method of breeding involves five or more generations of selfing andselection: S₁→S₂; S₂→S₃; S₃→S₄; S₄→S₅, etc. After at least fivegenerations, the inbred plant is considered genetically pure.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny are heterozygous for locicontrolling the characteristic being transferred, but are like thesuperior parent for most or almost all other loci. The last backcrossgeneration would be selfed to give pure breeding progeny for the traitbeing transferred.

The development of a hybrid corn variety involves three steps: (1) theselection of plants from various germplasm pools; (2) the selfing of theselected plants for several generations to produce a series of inbredplants, which, although different from each other, each breed true andare highly uniform; and (3) crossing the selected inbred plants withunrelated inbred plants to produce the hybrid progeny (F₁). During theinbreeding process in corn, the vigor of the plants decreases. Vigor isrestored when two unrelated inbred plants are crossed to produce thehybrid progeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred plants is that the hybrid between any twoinbreds is always the same. Once the inbreds that give a superior hybridhave been identified, hybrid seed can be reproduced indefinitely as longas the homogeneity of the inbred parents is maintained. Conversely, muchof the hybrid vigor exhibited by F₁ hybrids is lost in the nextgeneration (F₂). Consequently, seed from hybrid varieties is not usedfor planting stock. It is not generally beneficial for farmers to saveseed of F₁ hybrids. Rather, farmers purchase F₁ hybrid seed for plantingevery year.

The development of inbred plants generally requires at least about 5 to7 generations of selfing. Inbred plants are then cross-bred in anattempt to develop improved F₁ hybrids. Hybrids are then screened andevaluated in small scale field trials. Typically, about 10 to 15phenotypic traits, selected for their potential commercial value, aremeasured. A selection index of the most commercially important traits isused to help evaluate hybrids. FACT, an acronym for Field AnalysisComparison Trial (strip trials), is an on-farm experimental testingprogram employed by Monsanto Company to perform the final evaluation ofthe commercial potential of a product.

During the next several years, a progressive elimination of hybridsoccurs based on more detailed evaluation of their phenotype. Eventually,strip trials (FACT) are conducted to formally compare the experimentalhybrids being developed with other hybrids, some of which werepreviously developed and generally are commercially successful. That is,comparisons of experimental hybrids are made to competitive hybrids todetermine if there was any advantage to further development of theexperimental hybrids. Examples of such comparisons are presented hereinbelow. After FACT testing is complete, determinations may be madewhether commercial development should proceed for a given hybrid.

C. F₁ Hybrid Comparisons

As mentioned above, hybrids are progressively eliminated followingdetailed evaluations of their phenotype, including formal comparisonswith other commercially successful hybrids. Strip trials are used tocompare the phenotypes of hybrids grown in as many environments aspossible. They are performed in many environments to assess overallperformance of the new hybrids and to select optimum growing conditions.Because the corn is grown in close proximity, environmental factors thataffect gene expression, such as moisture, temperature, sunlight, andpests, are minimized. For a decision to be made to commercialize ahybrid, it is not necessary that the hybrid be better than all otherhybrids. Rather, significant improvements must be shown in at least sometraits that would create improvements in some niches.

Examples of such comparative data are set forth herein below in Table 2,which presents a comparison of performance data for a hybrid made withI135168 as one parent, versus selected hybrids of commercial value. Allthe data in Table 2 represents results across years and locations forresearch and/or strip trials.

TABLE 2 Comparative Data for NB2701, a Hybrid Having I135168 as OneInbred Parent SI YLD MST STL RTL SV TST HYBRID % C BU PTS % % RAT LBSNB2701 99.4 187.2 45.7 1.2 0.1 3.7 63.0 DKC27- 105.3 199.6 46.7 0.0 0.02.7 63.3 15 Diff −5.8 −12.4 −0.9 1.2 0.1 1.0 −0.3 p-value 0.442 0.2100.388 0.194 0.391 0.033 0.709 NB2701 99.4 187.2 57.0 1.2 0.1 4.2 63.0DKC27- 96.6 177.8 55.3 3.5 0.0 3.2 64.4 12 Diff 2.9 9.3 1.7 −2.2 0.1 1.0−1.3 p-value 0.537 0.270 0.181 0.003 0.391 0.001 0.184 LEGENDABBREVIATIONS: HYBD = Hybrid SI % C = Selection Index (percent of check)YLD BU/A = Yield (bushels/acre) MST PTS = Moisture STL % = Stalk Lodging(percent) RTL % = Root Lodging (percent) DRP % = Dropped Ears (percent)FLSTD % M = Final Stand (percent of test mean) SV RAT = Seedling VigorRating ELSTD % M = Early Stand (percent of test mean) PHT INCH = PlantHeight (inches) EHT INCH = Ear Height (inches) BAR % = Barren Plants(percent) SG RAT = Staygreen Rating TST LBS = Test Weight (pounds) FGDU= GDUs to Shed ESTR DAYS = Estimated Relative Maturity (days)

As can be seen in Table 2, the hybrid differs significantly in selectedtraits when compared to successful commercial hybrids.

D. Physical Description of F₁ Hybrids

The present invention provides F₁ hybrid corn plants derived from thecorn plant I135168. The physical characteristics of an exemplary hybridproduced using I135168 as one inbred parent are set forth in Table 3,which concerns NB2701. An explanation of terms used in Table 3 can befound in the Definitions, set forth hereinabove.

TABLE 3 Morphological Traits for NB2701, a Hybrid Having I135168 as OneInbred Parent CHARACTERISTIC VALUE 1. STALK Diameter (width) cm. 2.0Anthocyanin Absent Nodes With Brace Roots 1.2 Brace Root Color FaintInternode Direction Straight Internode Length cm. 16.7  2. LEAF ColorDark Green Length cm. 79.1  Width cm. 7.8 Sheath Anthocyanin AbsentSheath Pubescence Light Marginal Waves Moderate Longitudinal CreasesModerate 3. TASSEL Length cm. 41.7  Spike Length cm. 28.8  PeduncleLength cm. 4.8 Branch Number 4.6 Anther Color Salmon Glume Color LightRed Glume Band Absent 4. EAR Silk Color Tan Number Per Stalk 1.0Position (attitude) Upright Length cm. 17.8  Shape Semi-Conical Diametercm. 3.9 Shank Length cm. 9.6 Husk Bract Short Husk Cover cm. 0.4 HuskOpening Moderate Husk Color Fresh Green Husk Color Dry Buff Cob Diametercm. 2.0 Cob Color Red Shelling Percent 90.7  5. KERNEL Row Number 15.6 Number Per Row 35.2  Row Direction Straight Type Dent Cap Color YellowSide Color Deep Yellow Length (depth) mm. 11.0  Width mm. 8.0 Thickness4.6 Endosperm Type Normal Endosperm Color Yellow *These are typicalvalues. Values may vary due to environment. Other values that aresubstantially equivalent are also within the scope of the invention.VII. Genetic Complements

The present invention provides a genetic complement of the inbred cornplant variety designated I135168. Further provided by the invention is ahybrid genetic complement, wherein the complement is formed by thecombination of a haploid genetic complement from I135168 and anotherhaploid genetic complement. Means for determining such a geneticcomplement are well-known in the art.

As used herein, the phrase “genetic complement” means an aggregate ofnucleotide sequences, the expression of which defines the phenotype of acorn plant or a cell or tissue of that plant. By way of example, a cornplant is genotyped to determine a representative sample of the inheritedmarkers it possesses. Markers are alleles at a single locus. They arepreferably inherited in codominant fashion so that the presence of bothalleles at a diploid locus is readily detectable, and they are free ofenvironmental variation, i.e., their heritability is 1. This genotypingis preferably performed on at least one generation of the descendantplant for which the numerical value of the quantitative trait or traitsof interest are also determined. The array of single locus genotypes isexpressed as a profile of marker alleles, two at each locus. The markerallelic composition of each locus can be either homozygous orheterozygous. Homozygosity is a condition where both alleles at a locusare characterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

A genetic marker profile of an inbred may be predictive of the agronomictraits of a hybrid produced using that inbred. For example, if an inbredof known genetic marker profile and phenotype is crossed with a secondinbred of known genetic marker profile and phenotype it is possible topredict the phenotype of the F₁ hybrid based on the combined geneticmarker profiles of the parent inbreds. Methods for prediction of hybridperformance from genetic marker data is disclosed in U.S. Pat. No.5,492,547, the disclosure of which is specifically incorporated hereinby reference in its entirety. Such predictions may be made using anysuitable genetic marker, for example, SSRs, RFLPs, AFLPs, SNPs, orisozymes.

SSRs are genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can behighly informative in linkage analysis relative to other marker systemsin that multiple alleles may be present. Another advantage of this typeof marker is that, through use of flanking primers, detection of SSRscan be achieved, for example, by the polymerase chain reaction (PCR™),thereby eliminating the need for labor-intensive Southern hybridization.The PCR™ detection is done by use of two oligonucleotide primersflanking the polymorphic segment of repetitive DNA. Repeated cycles ofheat denaturation of the DNA followed by annealing of the primers totheir complementary sequences at low temperatures, and extension of theannealed primers with DNA polymerase, comprise the major part of themethodology. Following amplification, markers can be scored by gelelectrophoresis of the amplification products. Scoring of markergenotype is based on the size (number of base pairs) of the amplifiedsegment.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A seed of the corn variety I135168, wherein a representative sampleof seed of the variety has been deposited under ATCC AccessionNo.PTA-8160.
 2. A plant produced by growing the seed of claim
 1. 3. Aplant part of the plant of claim
 2. 4. The plant part of claim 3,further defined as pollen, a protoplast, an ovule or a cell.
 5. A tissueculture of cells obtained from the plant of claim
 2. 6. The tissueculture of claim 5, wherein the cells are from a tissue selected fromthe group consisting of leaf, pollen, embryo, root, root tip, anther,silk, flower, kernel, ear, cob, husk, stalk and meristem.
 7. A methodfor producing corn seed comprising growing the plant of claim 2 toproduce seed and collecting the seed.
 8. The method of claim 7, whereinthe corn seed is hybrid.
 9. The method of claim 7, wherein the seed isinbred seed.
 10. A seed produced by the method of claim
 7. 11. A methodof introducing a heritable trait selected from the group consisting ofmale sterility, herbicide tolerance, insect resistance, diseaseresistance, waxy starch, modified fatty acid metabolism, modified phyticacid metabolism, modified carbohydrate metabolism and modified proteinmetabolism into corn variety I135168 comprising the steps of: (a)crossing a plant of variety I135168, with another corn plant thatheritably carries the trait to produce F₁ progeny plants, at least someof which heritably carry the trait, wherein a representative sample ofseed of variety I135168 have been deposited under ATCC AccessionNo.PTA-8160; (b) selecting F₁ progeny plants that heritably carry thetrait; (c) crossing the selected progeny plants with another plant ofvariety I135168 to produce next-generation progeny plants at least someof which heritably carry the trait; (d) selecting next-generationprogeny plants that heritably carry the trait and exhibit themorphological and physiological characteristics of variety I135168; and(e) repeating steps (c) and (d) three or more times to produce selectedprogeny plants that comprise the trait and all of the morphological andphysical characteristics of corn variety I135168 set forth in Table 1 asdetermined at the 5% significance level when grown under the sameenvironmental conditions.
 12. A plant produced by the method of claim11, wherein the plant comprises the heritable trait and all of thephysiological and morphological characteristics of corn variety I135168listed in Table 1 as determined at the 5% significance level when grownunder the same environmental conditions.
 13. The plant of claim 12,wherein the heritable trait is herbicide tolerance and the tolerance isconferred to a herbicide selected from the group consisting of:imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 14. The plant of claim12, wherein the heritable trait is insect resistance and the insectresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 15. The plant of claim 12, wherein the heritable trait ismale sterility and the trait is conferred by a cytoplasmic nucleic acidmolecule that confers male sterility.
 16. The plant of claim 12, whereinthe heritable trait is modified fatty acid metabolism and the trait isconferred by a transgene encoding a fatty acid desaturase.
 17. The plantof claim 12, wherein the heritable trait is modified carbohydratemetabolism and the trait is conferred by a transgene encoding afructosyltransferase, levansucrase, alpha-amylase, invertase or starchbranching enzyme.
 18. A method of introducing a heritable trait selectedfrom the group consisting of male sterility, herbicide tolerance, insectresistance, disease resistance, waxy starch, modified fatty acidmetabolism, modified phytic acid metabolism, modified carbohydratemetabolism and modified protein metabolism into a plant of corn varietyI135168 comprising introducing a transgene conferring the trait into theplant, wherein a representative sample of seed of variety I135168 hasbeen deposited under ATCC Accession No.PTA-8160.
 19. A plant produced bythe method of claim 18, wherein the plant comprises the transgene andhas all of the physiological and morphological characteristics of cornvariety I135168 listed in Table 1 as determined at the 5% significancelevel when grown under the same environmental conditions.
 20. The plantof claim 19, wherein the heritable trait is herbicide tolerance and theresistance is conferred to an herbicide selected from the groupconsisting of: imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 21. The plant of claim19, wherein the heritable trait is insect resistance and the insectresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 22. The plant of claim 19, wherein the heritable trait ismodified fatty acid metabolism and the trait is conferred by a transgeneencoding a fatty acid desaturase.
 23. The plant of claim 19, wherein theheritable trait is modified carbohydrate metabolism and the trait isconferred by a transgene encoding a fructosyltransferase, levansucrase,alpha-amylase, invertase or starch branching enzyme.
 24. A method ofproducing an inbred corn plant derived from the corn variety I135168,the method comprising the steps of: (a) preparing a progeny plantderived from corn variety I135168 by crossing a plant of the cornvariety I135168 with a second corn plant, wherein a sample of the seedof the corn variety I135168 was deposited under ATCC AccessionNo.PTA-8160; (b) crossing the progeny plant with itself or a secondplant to produce a seed of a progeny plant of a subsequent generation;(c) growing a progeny plant of a subsequent generation from said seedand crossing the progeny plant of a subsequent generation with itself ora second plant; and (d) repeating steps (b) and (c) for an addition 3–10generations to produce an inbred corn plant derived from the cornvariety I135168.